Protective immunity to intracellular pathogens often requires the activity of cytotoxic T-lymphocytes (CTL). CTL classically recognize protein antigens produced in, or introduced into, the host cell cytosol. One challenge in stimulating CTL through immunization is directing the protein components of the vaccine into the host cell cytosol. We have developed a delivery system based on the ability of the protective antigen (PA) component of anthrax toxin to mediate entry of the toxin?s lethal factor (LF) and edema factor into the cytosolic compartment of mammalian cells. The amino-terminal domain of LF (LFn; 255 amino acids) is devoid of toxic activity and binds LF to PA. Heterologous proteins fused to LFn are delivered into the cytoplasm of host cells in the presence of PA. In the previous funding period we fused various pathogen-derived antigens to LFn and demonstrated the ability of the resulting LFn-antigen fusion proteins to stimulate CTL in mouse and human systems, and protect mice against challenge with model bacterial and viral pathogens. However, we have a limited understanding of how these fusion proteins generate protective immunity and whether the conditions we have used are optimal. In this submission we propose to: 1.) Investigate the magnitude and kinetics of antigen presentation in mice immunized with a toxin-epitope fusion protein, with the goal of improving the efficacy of vaccination with this system. 2.) Determine whether recognition of toxin-delivered antigen in the context of professional antigen presenting cells is required for stimulation of epitope-specific CTL. Treatment of professional antigen presenting cells with an AT fusion ex vivo and transfer of the treated cells into naive animals may generate a more potent CTL response that can be achieved by direct immunization. 3.) Investigate the use of an anthrax toxin-SIV fusion to stimulate protective CTL following immunization of rhesus macaques. The experiments proposed in these aims will yield insight into the mechanism by which these fusion proteins stimulate CTL and broaden the application of this approach. Expanding our understanding of how this vaccine system works should allow for its application in immunizing against a wide variety of bacterial, viral, and parasitic disease agents as well as in immunization or therapy of some cancers.